Liquid–liquid phase separation is a major mechanism of subcellular compartmentalization1,2. Although the segregation of RNA into phase-separated condensates broadly affects RNA metabolism3,4, whether and how specific RNAs use phase separation to regulate interacting factors such as RNA-binding proteins (RBPs), and the phenotypic consequences of such regulatory interactions, are poorly understood. Here we show that RNA-driven phase separation is a key mechanism through which a long noncoding RNA (lncRNA) controls the activity of RBPs and maintains genomic stability in mammalian cells. The lncRNA NORAD prevents aberrant mitosis by inhibiting Pumilio (PUM) proteins5,6,7,8. We show that NORAD can out-compete thousands of other PUM-binding transcripts to inhibit PUM by nucleating the formation of phase-separated PUM condensates, termed NP bodies. Dual mechanisms of PUM recruitment, involving multivalent PUM–NORAD and PUM–PUM interactions, enable NORAD to competitively sequester a super-stoichiometric amount of PUM in NP bodies. Disruption of NORAD-driven PUM phase separation leads to PUM hyperactivity and genome instability that is rescued by synthetic RNAs that induce the formation of PUM condensates. These results reveal a mechanism by which RNA-driven phase separation can regulate RBP activity and identify an essential role for this process in genome maintenance. The repetitive sequence architecture of NORAD and other lncRNAs9,10,11 suggests that phase separation may be a widely used mechanism of lncRNA-mediated regulation.
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All data needed to evaluate the conclusions of this Article are presented in the main text or supplementary materials. RNA-seq data for circPRE-expressing HCT116 cells (Fig. 4c) is available in the GEO under accession number GSE154812. HCT116 cell RNA-seq data used to estimate the total number of PREs expressed in mRNAs per cell is available in the GEO under accession number GSE75440. Source data are provided with this paper.
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We thank M. Rosen, D. Trono, S. Qi, J. Weissman, F. Zhang, X. Lian, and S. Jaffrey for plasmids; H. Zhang for bioinformatics support; S. Nakagawa for technical assistance with RNA FISH; M. Rosen, W. Peeples, J. Han, and B. Sabari for discussions; K. Jancynzka for experimental assistance; A. Mobley and UTSW Flow Cytometry Core for assistance with FACS; and K. O’Donnell, Y. Meleis, and members of the Mendell laboratory for comments on the manuscript. This work was supported by grants from the NIH (R35CA197311 to J.T.M.; P30CA142543 to J.T.M.; and P50CA196516 to J.T.M.) and the Welch Foundation (I-1961 to J.T.M.). J.T.M. is an Investigator of the Howard Hughes Medical Institute.
The authors declare no competing interests.
Peer review information Nature thanks Phillip A. Sharp and Igor Ulitsky for their contribution to the peer review of this work. Peer reviewer reports are available.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Left, confocal images of HCT116 cells co-stained for NORAD and PUM1 with or without treatment with camptothecin (Campto; 200 nM) for 24 h. Right, box plots of the mean fluorescence intensity and area of NORAD and PUM1 foci. NORAD, n = 149 foci; PUM1, n = 58 foci; NORAD + camptothecin, n = 97 foci; and PUM1 + camptothecin, n = 67 foci from at least two different fields analysed. Two-tailed t-test. b, Western blot of PUM1 or PUM2 in HCT116 cells of the indicated genotypes. Molecular weight in kDa shown on right. For gel source data, see Supplementary Fig. 1. c, Confocal images of PUM1 or PUM2 co-stained with P-body marker XRN1. d, Confocal images of PUM2 and stress granule marker G3BP1 with or without treatment with sodium arsenite (0.5 mM, 1 h incubation). e, Confocal images of NORAD and PUM localization in the indicated cell lines and genotypes. BJ-5ta cells are TERT-immortalized human fibroblasts. f, Quantification of NORAD and PUM1 co-localization (n = 20 cells for each cell line). Mean co-localization shown above box plots. a, f, Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum.
Extended Data Fig. 2 NP bodies are liquid-like condensates distinct from other known cytoplasmic granules.
a, PUM1 western blot in HCT116 clones with GFP knock-in at the endogenous PUM1 locus. Three genotype-confirmed wild-type and NORAD−/− clones shown alongside parental wild-type and NORAD−/− cells. Molecular weight in kDa shown on right. For gel source data, see Supplementary Fig. 1. b, Time-lapse images showing fusion of endogenous PUM1–GFP condensates in HCT116 cells. Scale bar, 5 μm. c, Representative 3D-reconstructed live cell confocal images used to estimate the volumes of NP bodies, P-bodies, and stress granules. Endogenously tagged PUM1–GFP HCT116 cells were used for NP body and stress granule measurements (the latter after treatment with 0.5 mM sodium arsenite for 1 h). The P-body marker DCP2–GFP was used to estimate the volume of P-bodies in HCT116 cells. d, Quantification of condensate volumes (n = 1,072 NP bodies from 20 cells, 51 P-bodies from 11 cells, and 62 stress granules from 10 cells). Average volumes shown above plots.
a, Predicted disordered regions of PUM1 and PUM2, scored by PONDR VSL2. b, Schematic (top) and Coomassie stain (bottom) of purified MBP–SNAP-PUM–His proteins used for in vitro experiments. For gel source data, see Supplementary Fig. 1. c, PUM liquid droplet formation at 5 μM upon TEV-mediated cleavage of the solubilizing MBP tag. Proteins were fluorescently labelled with SNAP488 and visualized by DIC or fluorescence microscopy. Scale bar, 10 μm. d, PUM1 liquid droplet formation at 5 μM in the presence or absence of 10% PEG3350 after 1 h incubation. e, Confocal images (left) and quantification (right) of PUM1 droplet FRAP (5 μM PUM1). Fluorescence intensities plotted relative to pre-bleach time point (t = −5 s). Data shown as mean ± s.d. (n = 3 droplets). f, Time-lapse confocal images showing fusion of PUM1 droplets (5 μM PUM1). g, DIC and fluorescence microscopy images of Cy3-labelled NORAD RNA (2.7 nM, red) after addition to preformed PUM1 or PUM2 droplets (5 μM, green). h, Fluorescence microscopy images of PUM1 droplets (5 μM, green) formed in the presence or absence of NORAD or PREmut RNA (2.7 nM, red). PREmut contains UGU to ACA mutations in all 18 NORAD PREs, which abolish PUM binding5. i, Images and analysis of PUM1–NORAD droplet FRAP. Droplets were formed with Cy3-labelled NORAD RNA (2 nM) and SNAP488-labelled PUM1 (5 μM). Data shown as mean ± s.d. (n = 3 droplets).
a, Representative 3D-reconstructed confocal image of a PUM1-stained NORAD−/− HCT116 cell used to estimate cytoplasmic volume. The length (l), width (w), and depth (d) of the entire cell and its nucleus were measured and used to calculate the total cellular and nuclear volumes using the ellipsoid volume formula. Cytoplasmic volume for each cell was determined by subtracting nuclear volume from total volume. b, Box-and-whisker plot of measured cytoplasmic volumes (n = 20 cells). Mean cytoplasmic volume shown on the right. Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum. c, Schematic of wild-type and mutant NORAD transcripts. Location of repeated NORAD domains (ND1–ND5) indicated with grey boxes and mammalian sequence conservation shown in green (UCSC Genome Browser hg38 PhastCons track). Locations of PREs indicated with red and yellow arrowheads. PREmut transcript contains 18 UGU to ACA mutations in PREs (grey arrowheads). ND4 represents the most conserved segment of NORAD and contains 4 PREs. Figure modified from Elguindy et al.5. d, DIC and fluorescence microscopy images of PUM1 (150 nM, green) droplets in the presence or absence of NORAD (2 nM) or PEG3350.
Extended Data Fig. 5 PUM target transcripts do not induce PUM1 droplet formation in vitro at physiological concentrations.
a, Dot plot of PRE-containing mRNA copy numbers in HCT116 cells (estimated by RNA-seq; see Methods) versus the number of PREs in their 3′ UTRs. Transcripts selected for in vitro assays labelled in blue with the number of PREs in their 3′ UTRs in parentheses. Note that the selected mRNAs were also identified as PUM CLIP targets that were downregulated upon PUM overexpression7,39. b, Plot of transcript PRE number multiplied by estimated copy number, highlighting the unique combination of NORAD abundance and PRE valency compared to other PRE-containing RNAs. c, qRT–PCR validation of copy numbers of the indicated transcripts in HCT116 cells. Mean copy number is shown above each bar. Data shown as mean ± s.d. n = 3 biological replicates. d, Approximate cytoplasmic concentration of each PUM target transcript in HCT116 cells. e, Confocal images of PUM1 droplets, formed with 150 nM PUM1 plus 2 nM NORAD or increasing concentrations of the indicated in vitro transcribed 3′ UTR of each PUM target. Red boxes highlight assays performed at the estimated physiologic concentration of each transcript in HCT116 cells. The same PUM1 protein preparation was used for droplet formation assays with NORAD and 3′ UTRs.
a, Confocal images of HCT116 cells co-stained for the indicated RNA and PUM1 by RNA FISH and immunofluorescence, respectively. b, Quantification of the indicated PUM target mRNA and PUM1 co-localization (n = 20 cells for each RNA). Mean co-localization shown above box plots. Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum co-localization for each target mRNA.
Extended Data Fig. 7 Competitive, super-stoichiometric recruitment of PUM into RNA-induced droplets.
a, Quantification of the number of PUM1 molecules per PRE in droplets nucleated by NORAD (2 nM) or PRE8 oligonucleotide (10 nM) and the indicated concentration of PUM1 in vitro. Black line represents the mean (n = 101, 115, 113, 107, 114, and 115 droplets for each condition from left to right). b, Quantification of PUM1 partition coefficients formed with 150 nM PUM1 and 10 nM PRE8 RNA normalized to partition coefficient at 0 μM competitor RNA (PRE1 RNA). IC50 represents concentration of PRE1 RNA needed to reduce PUM1 partitioning by 50%. Data shown as mean ± s.d. n = 44 or more droplets analysed for each data point. Each PRE in the PRE8 RNA is approximately 9 times more efficient at PUM1 recruitment than a monovalent PRE.
a, Structure of the human PUM1 HD domain (PDB: 1M8X) in complex with PRE–RNA, showing mutated residues in PUM1HDmut (pink). b, EMSA demonstrating loss of PUM1HDmut RNA binding. c, Coomassie stain of purified MBP–SNAP–PUM1HDmut–His protein used for in vitro experiments. d, Droplet formation by the indicated PUM proteins in the presence or absence of PRE8 RNA oligonucleotide. e, Coomassie stain of purified MBP–SNAP–PUM1HD-WTΔIDR–His and MBP–SNAP–PUM1HDmutΔIDR–His proteins used for in vitro experiments. Gels were cropped to remove irrelevant lanes where indicated with vertical lines. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 9 Recruitment of PUM proteins into pre-formed NORAD–PUM condensates independently of RNA binding.
a, Western blot analysis of PUM1 (left) and PUM2 (right) in HCT116 cells of the indicated genotypes transduced with lentiviruses expressing PUMWT–GFP or PUMHDmut–GFP. Molecular weight in kDa shown on right. For gel source data, see Supplementary Fig. 1. b, Time-lapse live-cell confocal images showing fusion of PUM1WT–GFP or PUM1HDmut–GFP condensates in wild-type HCT116 cells. Scale bar, 5 μm. c, d, Top, images of PUM1WT–GFP and PUM1HDmut–GFP FRAP (c) or PUM2WT–GFP and PUM2HDmut–GFP FRAP (d) in wild-type HCT116 cells. Puncta undergoing photobleaching shown in dashed boxes. Bottom, FRAP quantification with fluorescence intensities plotted relative to pre-bleach time point (t = –5 s). Data shown as mean ± s.d. (c; n = 3 puncta) or mean (d; n = 2 puncta). Scale bar, 5 μm. e, Confocal images of PUM2WT–GFP (top) and PUM2HDmut–GFP (bottom) in HCT116 cells of the indicated genotypes. f, Left, confocal images of GFP-tagged full-length or IDR-deleted PUM1WT or PUM1HDmut expressed in wild-type HCT116 cells. Right, quantification of PUM1 partition coefficients, defined as the intensity of PUM1–GFP inside condensates relative to the surrounding cytoplasm. Partition coefficients were calculated for n = 175, 116, 36, or 121 condensates from 5 different cells for each protein from left to right. Black bar depicts the mean partition coefficient. Two-tailed t-test comparing each PUM1 mutant to PUM1WT. ****P < 1 × 10−15; n.s., not significant.
a, Schematic of circPRE-producing constructs21, which encode the Broccoli aptamer and 0–8 PREs. b, Copy number analysis of circPRE-transcripts in HCT116 CRISPRi cells expressing control or NORAD-targeting sgRNAs. Mean copy number in sgNORAD cell lines is shown above each bar. circPRE4-low and circPRE4-mid represent distinct cell populations sorted for different circPRE copy numbers. Data shown as mean ± s.d. n = 3 biological replicates. c, qRT–PCR analysis of NORAD expression in the indicated circPRE HCT116 CRISPRi cell lines expressing control or NORAD-targeting sgRNAs. Data shown as mean ± s.d. n = 3 technical replicates. d, e, Top, confocal images of PUM1 (d) or PUM2 (e) immunofluorescence in the indicated cell lines. Bottom, quantification of the number of PUM1 (d) or PUM2 (e) foci per cell in the indicated sgControl- or sgNORAD-infected cell lines (n = 20 cells for each cell line). Mean number of foci shown above each box plot. Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum.
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Elguindy, M.M., Mendell, J.T. NORAD-induced Pumilio phase separation is required for genome stability. Nature 595, 303–308 (2021). https://doi.org/10.1038/s41586-021-03633-w